The goal of this proposal is to identify cellular processes and gene expression patterns regulated by normal and oncogenic forms of TFE3, a transcription factor involved in papillary renal cell carcinoma (PRCC) and TGFb responsiveness. TFE3, a member of the MiT transcription factor family that includes Mitf, the gene mutated in microphthalmia, has been implicated in TGFa, Wnt, and c-kit signaling pathways. Our preliminary experiments indicate that ectopic expression of TFE3, but not other MiT proteins, triggers apoptotic cell death, implicating TFE3 in cell cycle control. Translocations of the TFE3 gene are found in a subset of sporadic juvenile forms of PRCC, supporting a key role for TFE3 mutation in transformation. Four distinct TFE3 translocation partners have been identified, creating fusion proteins linking the C-terminal portion of TFE3, which contains the DNA binding, dimerization, and a transcriptional activation domain, with the other gene product. The molecular basis for how these chimeric proteins contribute to the transformation process of renal epithelial cells, and the normal function of TFE3 in the kidney and in cytokine responsiveness, are not known. We propose in Aim 1 to identify and characterize the activity of MiT proteins, including TFE3, in renal epithelial cells. Towards this goal, we have created a transdominant negative (TDN) protein that specifically blocks the activity of all MiT family members. In Aim 2, we will monitor the impact of the TDN and TFE3 oncoprotein expression on cytokine responsiveness of renal epithelial cells. As part of this aim, we will test the hypothesis that TFE3 is a mediator of c-met signaling; c-met, the hepatocyte growth factor (HGF) receptor, is mutated in a subset of hereditary PRCC. In Aim 3, we will identify portions and activities of TFE3 responsible for apoptosis. In Aim 4, we will express the TDN, oncoproteins, and normal TFE3 proteins in renal epithelial and non-kidney cells for comparative gene expression analyses to identify target genes regulated by the TFE3 proteins. Included will be the evaluation of TAT-TDN, a fusion of the HIV TAT protein to the TDN protein. This will allow the TDN to autonomously permeate the cell membrane and enable us to inhibit TFE3 activity in quiescent cells. An important outcome will be to establish the ability of the TDN to block TFE3 oncoprotein activity. Our studies will enhance our understanding of how TFE3 mutation promotes transformation of renal cells, and provide insight into new interventive strategies for PRCC.